Chemoproteomic discovery of a covalent allosteric inhibitor of WRN helicase.

WRN helicase is a promising target for treatment of cancers with microsatellite instability (MSI) due to its essential role in resolving deleterious non-canonical DNA structures that accumulate in cells with faulty mismatch repair mechanisms1-5. Currently there are no approved drugs directly targeting human DNA or RNA helicases, in part owing to the challenging nature of developing potent and selective compounds to this class of proteins. Here we describe the chemoproteomics-enabled discovery of a clinical-stage, covalent allosteric inhibitor of WRN, VVD-133214. This compound selectively engages a cysteine (C727) located in a region of the helicase domain subject to interdomain movement during DNA unwinding. VVD-133214 binds WRN protein cooperatively with nucleotide and stabilizes compact conformations lacking the dynamic flexibility necessary for proper helicase function, resulting in widespread double-stranded DNA breaks, nuclear swelling and cell death in MSI-high (MSI-H), but not in microsatellite-stable, cells. The compound was well tolerated in mice and led to robust tumour regression in multiple MSI-H colorectal cancer cell lines and patient-derived xenograft models. Our work shows an allosteric approach for inhibition of WRN function that circumvents competition from an endogenous ATP cofactor in cancer cells, and designates VVD-133214 as a promising drug candidate for patients with MSI-H cancers.

Discovery of Orally Available and Brain Penetrant AEP Inhibitors.

Alzheimer's Disease (AD) is the most widespread form of dementia, with one of the pathological hallmarks being the formation of neurofibrillary tangles (NFTs). These tangles consist of phosphorylated Tau fragments. Asparagine endopeptidase (AEP) is a key Tau cleaving enzyme that generates aggregation-prone Tau fragments. Inhibition of AEP to reduce the level of toxic Tau fragment formation could represent a promising therapeutic strategy. Here, we report the first orthosteric, selective, orally bioavailable, and brain penetrant inhibitors with an irreversible binding mode. We outline the development of the series starting from reversible molecules and demonstrate the link between inhibition of AEP and reduction of Tau N368 fragment both in vitro and in vivo.

Details Matter in Structure-based Drug Design.

Successful structure-based drug design (SBDD) requires the optimization of interactions with the target protein and the minimization of ligand strain. Both factors are often modulated by small changes in the chemical structure which can lead to profound changes in the preferred conformation and interaction preferences of the ligand. We draw from examples of a Roche project targeting phosphodiesterase 10 to highlight that details matter in SBDD. Data mining in crystal structure databases can help to identify these sometimes subtle effects, but it is also a great resource to learn about molecular recognition in general and can be used as part of molecular design tools. We illustrate the use of the Cambridge Structural Database for identifying preferred structural motifs for intramolecular hydrogen bonding and of the Protein Data Bank for deriving propensities for protein-ligand interactions.

Discovery of a Series of Indane-Containing NBTIs with Activity against Multidrug-Resistant Gram-Negative Pathogens.

The rise of multidrug-resistant (MDR) Gram-negative bacteria is a major global health problem necessitating the discovery of new classes of antibiotics. Novel bacterial topoisomerase inhibitors (NBTIs) target the clinically validated bacterial type II topoisomerases with a distinct binding site and mechanism of action to fluoroquinolone antibiotics, thus avoiding cross-resistance to this drug class. Here we report the discovery of a series of NBTIs incorporating a novel indane DNA binding moiety. X-ray cocrystal structures of compounds 2 and 17a bound to Staphylococcus aureus DNA gyrase-DNA were determined, revealing specific interactions with the enzyme binding pocket at the GyrA dimer interface and a long-range electrostatic interaction between the basic amine in the linker and the carboxylate of Asp83. Exploration of the structure-activity relationship within the series led to the identification of lead compound 18c, which showed potent broad-spectrum activity against a panel of MDR Gram-negative bacteria.

Structure of reverse gyrase with a minimal latch that supports ATP-dependent positive supercoiling without specific interactions with the topoisomerase domain.

Reverse gyrase is the only topoisomerase that introduces positive supercoils into DNA in an ATP-dependent reaction. Positive DNA supercoiling becomes possible through the functional cooperation of the N-terminal helicase domain of reverse gyrase with its C-terminal type IA topoisomerase domain. This cooperation is mediated by a reverse-gyrase-specific insertion into the helicase domain termed the `latch'. The latch consists of a globular domain inserted at the top of a ß-bulge loop that connects this globular part to the helicase domain. While the globular domain shows little conservation in sequence and length and is dispensable for DNA supercoiling, the ß-bulge loop is required for supercoiling activity. It has previously been shown that the ß-bulge loop constitutes a minimal latch that couples ATP-dependent processes in the helicase domain to DNA processing by the topoisomerase domain. Here, the crystal structure of Thermotoga maritima reverse gyrase with such a ß-bulge loop as a minimal latch is reported. It is shown that the ß-bulge loop supports ATP-dependent DNA supercoiling of reverse gyrase without engaging in specific interactions with the topoisomerase domain. When only a small latch or no latch is present, a helix in the nearby helicase domain of T. maritima reverse gyrase partially unfolds. Comparison of the sequences and predicted structures of latch regions in other reverse gyrases shows that neither sequence nor structure are decisive factors for latch functionality; instead, the decisive factors are likely to be electrostatics and plain steric bulk.

A high quality, industrial data set for binding affinity prediction: performance comparison in different early drug discovery scenarios.

We release a new, high quality data set of 1162 PDE10A inhibitors with experimentally determined binding affinities together with 77 PDE10A X-ray co-crystal structures from a Roche legacy project. This data set is used to compare the performance of different 2D- and 3D-machine learning (ML) as well as empirical scoring functions for predicting binding affinities with high throughput. We simulate use cases that are relevant in the lead optimization phase of early drug discovery. ML methods perform well at interpolation, but poorly in extrapolation scenarios-which are most relevant to a real-world application. Moreover, we find that investing into the docking workflow for binding pose generation using multi-template docking is rewarded with an improved scoring performance. A combination of 2D-ML and 3D scoring using a modified piecewise linear potential shows best overall performance, combining information on the protein environment with learning from existing SAR data.

Synthesis, Characterization, and in vivo Evaluation of a Novel Potent Autotaxin-Inhibitor.

The autotaxin-lysophosphatidic acid (ATX-LPA) signaling pathway plays a role in a variety of autoimmune diseases, such as rheumatoid arthritis or neurodegeneration. A link to the pathogenesis of glaucoma is suggested by an overactive ATX-LPA axis in aqueous humor samples of glaucoma patients. Analysis of such samples suggests that the ATX-LPA axis contributes to the fibrogenic activity and resistance to aqueous humor outflow through the trabecular meshwork. In order to inhibit or modulate this pathway, we developed a new series of ATX-inhibitors containing novel bicyclic and spirocyclic structural motifs. A potent lead compound (IC50 against ATX: 6 nM) with good in vivo PK, favorable in vitro property, and safety profile was generated. This compound leads to lowered LPA levels in vivo after oral administration. Hence, it was suitable for chronic oral treatment in two rodent models of glaucoma, the experimental autoimmune glaucoma (EAG) and the ischemia/reperfusion models. In the EAG model, rats were immunized with an optic nerve antigen homogenate, while controls received sodium chloride. Retinal ischemia/reperfusion (I/R) was induced by elevating the intraocular pressure (IOP) in one eye to 140 mmHg for 60 min, followed by reperfusion, while the other untreated eye served as control. Retinae and optic nerves were evaluated 28 days after EAG or 7 and 14 days after I/R induction. Oral treatment with the optimized ATX-inhibitor lead to reduced retinal ganglion cell (RGC) loss in both glaucoma models. In the optic nerve, the protective effect of ATX inhibition was less effective compared to the retina and only a trend to a weakened neurofilament distortion was detectable. Taken together, these results provide evidence that the dysregulation of the ATX-LPA axis in the aqueous humor of glaucoma patients, in addition to the postulated outflow impairment, might also contribute to RGC loss. The observation that ATX-inhibitor treatment in both glaucoma models did not result in significant IOP increases or decreases after oral treatment indicates that protection from RGC loss due to inhibition of the ATX-LPA axis is independent of an IOP lowering effect.

Small molecule AX-024 reduces T cell proliferation independently of CD3ϵ/Nck1 interaction, which is governed by a domain swap in the Nck1-SH3.1 domain.

Activation of the T cell receptor (TCR) results in binding of the adapter protein Nck (noncatalytic region of tyrosine kinase) to the CD3ϵ subunit of the TCR. The interaction was suggested to be important for the amplification of TCR signals and is governed by a proline-rich sequence (PRS) in CD3ϵ that binds to the first Src homology 3 (SH3) domain of Nck (Nck-SH3.1). Inhibition of this protein/protein interaction ameliorated inflammatory symptoms in mouse models of multiple sclerosis, psoriasis, and asthma. A small molecule, AX-024, was reported to inhibit the Nck/CD3ϵ interaction by physically binding to the Nck1-SH3.1 domain, suggesting a route to develop an inhibitor of the Nck1/CD3ϵ interaction for modulating TCR activity in autoimmune and inflammatory diseases. We show here that AX-024 reduces T cell proliferation upon weak TCR stimulation but does not significantly affect phosphorylation of Zap70 (ζ chain of T cell receptor-associated protein kinase 70). We also find that AX-024 is likely not involved in modulating the Nck/TCR interaction but probably has other targets in T cells. An array of biophysical techniques did not detect a direct interaction between AX-024 and Nck-SH3.1 in vitro Crystal structures of the Nck-SH3.1 domain revealed its binding mode to the PRS in CD3ϵ. The SH3 domain tends to generate homodimers through a domain swap. Domain swaps observed previously in other SH3 domains indicate a general propensity of this protein fold to exchange structural elements. The swapped form of Nck-SH3.1 is unable to bind CD3ϵ, possibly representing an inactive form of Nck in cells.

Structural Basis for Allosteric Ligand Recognition in the Human CC Chemokine Receptor 7.

The CC chemokine receptor 7 (CCR7) balances immunity and tolerance by homeostatic trafficking of immune cells. In cancer, CCR7-mediated trafficking leads to lymph node metastasis, suggesting the receptor as a promising therapeutic target. Here, we present the crystal structure of human CCR7 fused to the protein Sialidase NanA by using data up to 2.1 Å resolution. The structure shows the ligand Cmp2105 bound to an intracellular allosteric binding pocket. A sulfonamide group, characteristic for various chemokine receptor ligands, binds to a patch of conserved residues in the Gi protein binding region between transmembrane helix 7 and helix 8. We demonstrate how structural data can be used in combination with a compound repository and automated thermal stability screening to identify and modulate allosteric chemokine receptor antagonists. We detect both novel (CS-1 and CS-2) and clinically relevant (CXCR1-CXCR2 phase-II antagonist Navarixin) CCR7 modulators with implications for multi-target strategies against cancer.

DNA-Encoded Library-Derived DDR1 Inhibitor Prevents Fibrosis and Renal Function Loss in a Genetic Mouse Model of Alport Syndrome.

The importance of Discoidin Domain Receptor 1 (DDR1) in renal fibrosis has been shown via gene knockout and use of antisense oligonucleotides; however, these techniques act via a reduction of DDR1 protein, while we prove the therapeutic potential of inhibiting DDR1 phosphorylation with a small molecule. To date, efforts to generate a selective small-molecule to specifically modulate the activity of DDR1 in an in vivo model have been unsuccessful. We performed parallel DNA encoded library screens against DDR1 and DDR2, and discovered a chemical series that is highly selective for DDR1 over DDR2. Structure-guided optimization efforts yielded the potent DDR1 inhibitor 2.45, which possesses excellent kinome selectivity (including 64-fold selectivity over DDR2 in a biochemical assay), a clean in vitro safety profile, and favorable pharmacokinetic and physicochemical properties. As desired, compound 2.45 modulates DDR1 phosphorylation in vitro as well as prevents collagen-induced activation of renal epithelial cells expressing DDR1. Compound 2.45 preserves renal function and reduces tissue damage in Col4a3-/- mice (the preclinical mouse model of Alport syndrome) when employing a therapeutic dosing regime, indicating the real therapeutic value of selectively inhibiting DDR1 phosphorylation in vivo. Our results may have wider significance as Col4a3-/- mice also represent a model for chronic kidney disease, a disease which affects 10% of the global population.

Domain swap in the C-terminal ubiquitin-like domain of human doublecortin.

Doublecortin, a microtubule-associated protein that is only produced during neurogenesis, cooperatively binds to microtubules and stimulates microtubule polymerization and cross-linking by unknown mechanisms. A domain swap is observed in the crystal structure of the C-terminal domain of doublecortin. As determined by analytical ultracentrifugation, an open conformation is also present in solution. At higher concentrations, higher-order oligomers of the domain are formed. The domain swap and additional interfaces observed in the crystal lattice can explain the formation of doublecortin tetramers or multimers, in line with the analytical ultracentrifugation data. Taken together, the domain swap offers a mechanism for the observed cooperative binding of doublecortin to microtubules. Doublecortin-induced cross-linking of microtubules can be explained by the same mechanism. The effect of several mutations leading to lissencephaly and double-cortex syndrome can be traced to the domain swap and the proposed self-association of doublecortin.

Ligand channel in pharmacologically stabilized rhodopsin.

In the degenerative eye disease retinitis pigmentosa (RP), protein misfolding leads to fatal consequences for cell metabolism and rod and cone cell survival. To stop disease progression, a therapeutic approach focuses on stabilizing inherited protein mutants of the G protein-coupled receptor (GPCR) rhodopsin using pharmacological chaperones (PC) that improve receptor folding and trafficking. In this study, we discovered stabilizing nonretinal small molecules by virtual and thermofluor screening and determined the crystal structure of pharmacologically stabilized opsin at 2.4 Å resolution using one of the stabilizing hits (S-RS1). Chemical modification of S-RS1 and further structural analysis revealed the core binding motif of this class of rhodopsin stabilizers bound at the orthosteric binding site. Furthermore, previously unobserved conformational changes are visible at the intradiscal side of the seven-transmembrane helix bundle. A hallmark of this conformation is an open channel connecting the ligand binding site with the membrane and the intradiscal lumen of rod outer segments. Sufficient in size, the passage permits the exchange of hydrophobic ligands such as retinal. The results broaden our understanding of rhodopsin's conformational flexibility and enable therapeutic drug intervention against rhodopsin-related retinitis pigmentosa.

D3R Grand Challenge 2: blind prediction of protein-ligand poses, affinity rankings, and relative binding free energies.

The Drug Design Data Resource (D3R) ran Grand Challenge 2 (GC2) from September 2016 through February 2017. This challenge was based on a dataset of structures and affinities for the nuclear receptor farnesoid X receptor (FXR), contributed by F. Hoffmann-La Roche. The dataset contained 102 IC50 values, spanning six orders of magnitude, and 36 high-resolution co-crystal structures with representatives of four major ligand classes. Strong global participation was evident, with 49 participants submitting 262 prediction submission packages in total. Procedurally, GC2 mimicked Grand Challenge 2015 (GC2015), with a Stage 1 subchallenge testing ligand pose prediction methods and ranking and scoring methods, and a Stage 2 subchallenge testing only ligand ranking and scoring methods after the release of all blinded co-crystal structures. Two smaller curated sets of 18 and 15 ligands were developed to test alchemical free energy methods. This overview summarizes all aspects of GC2, including the dataset details, challenge procedures, and participant results. We also consider implications for progress in the field, while highlighting methodological areas that merit continued development. Similar to GC2015, the outcome of GC2 underscores the pressing need for methods development in pose prediction, particularly for ligand scaffolds not currently represented in the Protein Data Bank ( http://www.pdb.org ), and in affinity ranking and scoring of bound ligands.

Discovery of a microbial transglutaminase enabling highly site-specific labeling of proteins.

Microbial transglutaminases (MTGs) catalyze the formation of Gln-Lys isopeptide bonds and are widely used for the cross-linking of proteins and peptides in food and biotechnological applications (e.g. to improve the texture of protein-rich foods or in generating antibody-drug conjugates). Currently used MTGs have low substrate specificity, impeding their biotechnological use as enzymes that do not cross-react with nontarget substrates (i.e. as bio-orthogonal labeling systems). Here, we report the discovery of an MTG from Kutzneria albida (KalbTG), which exhibited no cross-reactivity with known MTG substrates or commonly used target proteins, such as antibodies. KalbTG was produced in Escherichia coli as soluble and active enzyme in the presence of its natural inhibitor ammonium to prevent potentially toxic cross-linking activity. The crystal structure of KalbTG revealed a conserved core similar to other MTGs but very short surface loops, making it the smallest MTG characterized to date. Ultra-dense peptide array technology involving a pool of 1.4 million unique peptides identified specific recognition motifs for KalbTG in these peptides. We determined that the motifs YRYRQ and RYESK are the best Gln and Lys substrates of KalbTG, respectively. By first reacting a bifunctionalized peptide with the more specific KalbTG and in a second step with the less specific MTG from Streptomyces mobaraensis, a successful bio-orthogonal labeling system was demonstrated. Fusing the KalbTG recognition motif to an antibody allowed for site-specific and ratio-controlled labeling using low label excess. Its site specificity, favorable kinetics, ease of use, and cost-effective production render KalbTG an attractive tool for a broad range of applications, including production of therapeutic antibody-drug conjugates.

Structure of the malaria vaccine candidate antigen CyRPA and its complex with a parasite invasion inhibitory antibody.

Invasion of erythrocytes by Plasmodial merozoites is a composite process involving the interplay of several proteins. Among them, the Plasmodium falciparum Cysteine-Rich Protective Antigen (PfCyRPA) is a crucial component of a ternary complex, including Reticulocyte binding-like Homologous protein 5 (PfRH5) and the RH5-interacting protein (PfRipr), essential for erythrocyte invasion. Here, we present the crystal structures of PfCyRPA and its complex with the antigen-binding fragment of a parasite growth inhibitory antibody. PfCyRPA adopts a 6-bladed ß-propeller structure with similarity to the classic sialidase fold, but it has no sialidase activity and fulfills a purely non-enzymatic function. Characterization of the epitope recognized by protective antibodies may facilitate design of peptidomimetics to focus vaccine responses on protective epitopes. Both in vitro and in vivo anti-PfCyRPA and anti-PfRH5 antibodies showed more potent parasite growth inhibitory activity in combination than on their own, supporting a combined delivery of PfCyRPA and PfRH5 in vaccines.

Quadruple space-group ambiguity owing to rotational and translational noncrystallographic symmetry in human liver fructose-1,6-bisphosphatase.

Fructose-1,6-bisphosphatase (FBPase) is a key regulator of gluconeogenesis and a potential drug target for type 2 diabetes. FBPase is a homotetramer of 222 symmetry with a major and a minor dimer interface. The dimers connected via the minor interface can rotate with respect to each other, leading to the inactive T-state and active R-state conformations of FBPase. Here, the first crystal structure of human liver FBPase in the R-state conformation is presented, determined at a resolution of 2.2 Šin a tetragonal setting that exhibits an unusual arrangement of noncrystallographic symmetry (NCS) elements. Self-Patterson function analysis and various intensity statistics revealed the presence of pseudo-translation and the absence of twinning. The space group is P41212, but structure determination was also possible in space groups P43212, P4122 and P4322. All solutions have the same arrangement of three C2-symmetric dimers spaced by 1/3 along an NCS axis parallel to the c axis located at (1/4, 1/4, z), which is therefore invisible in a self-rotation function analysis. The solutions in the four space groups are related to one another and emulate a body-centred lattice. If all NCS elements were crystallographic, the space group would be I4122 with a c axis three times shorter and a single FBPase subunit in the asymmetric unit. I4122 is a minimal, non-isomorphic supergroup of the four primitive tetragonal space groups, explaining the space-group ambiguity for this crystal.

Design and synthesis of selective, dual fatty acid binding protein 4 and 5 inhibitors.

Dual inhibition of fatty acid binding proteins 4 and 5 (FABP4 and FABP5) is expected to provide beneficial effects on a number of metabolic parameters such as insulin sensitivity and blood glucose levels and should protect against atherosclerosis. Starting from a FABP4 selective focused screening hit, biostructure information was used to modulate the selectivity profile in the desired way and to design potent dual FABP4/5 inhibitors with good selectivity against FABP3. With very good pharmacokinetic properties and no major safety alerts, compound 12 was identified as a suitable tool compound for further in vivo investigations.

Crystal Structures of the Human Doublecortin C- and N-terminal Domains in Complex with Specific Antibodies.

Doublecortin is a microtubule-associated protein produced during neurogenesis. The protein stabilizes microtubules and stimulates their polymerization, which allows migration of immature neurons to their designated location in the brain. Mutations in the gene that impair doublecortin function and cause severe brain formation disorders are located on a tandem repeat of two doublecortin domains. The molecular mechanism of action of doublecortin is only incompletely understood. Anti-doublecortin antibodies, such as the rabbit polyclonal Abcam 18732, are widely used as neurogenesis markers. Here, we report the generation and characterization of antibodies that bind to single doublecortin domains. The antibodies were used as tools to obtain structures of both domains. Four independent crystal structures of the N-terminal domain reveal several distinct open and closed conformations of the peptide linking N- and C-terminal domains, which can be related to doublecortin function. An NMR assignment and a crystal structure in complex with a camelid antibody fragment show that the doublecortin C-terminal domain adopts the same well defined ubiquitin-like fold as the N-terminal domain, despite its reported aggregation and molten globule-like properties. The antibodies' unique domain specificity also renders them ideal research tools to better understand the role of individual domains in doublecortin function. A single chain camelid antibody fragment specific for the C-terminal doublecortin domain affected microtubule binding, whereas a monoclonal mouse antibody specific for the N-terminal domain did not. Together with steric considerations, this suggests that the microtubule-interacting doublecortin domain observed in cryo-electron micrographs is the C-terminal domain rather than the N-terminal one.